Characterizing the ligand specificity of the RNA binding protein LARP6
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The La-related proteins are a superfamily of RNA-binding proteins characterized by a unique RNA binding domain that is comprised of two subdomains, the La motif (LAM) and the RNA recognition motif (RRM), which are connected by a flexible linker. It is still unclear whether ligand recognition by LARP6 is sequence-specific or dependent on formation of specific three-dimensional structures. The lack of a clear binding mechanism by LARP6 limits the ability of the field to identify other ligands for this protein. The LARP6 protein is only known to bind to a hairpin structure that is found in the 5’ untranslated region (UTR) of collagen type I mRNA (COL1A1). Interestingly, a similar hairpin structure with a high degree of sequence conservation is found in the 5’UTRs of three other fibrillar human collagen mRNAs (COL1A2, COL3A1 and COL5A2). The goal of this work was to determine the affinity and specificity of LARP6 binding to this set of collagen mRNAs. To accomplish this, we first optimized a native electrophoretic mobility shift assay (EMSA) using biotinylated ligands. Using this system, we have shown that LARP6 binds with high affinity (in the low nanomolar range) to all of the collagen sequences. Unexpectedly, LARP6 was also observed to bind with similar affinity to a random RNA sequence with a predicted secondary structure very different from the collagen mRNA ligands. These data suggest that LARP6 is able to bind RNAs due to nonspecific ionic interactions with the RNA phosphate backbone. This hypothesis was tested by evaluating the dependence of the apparent binding affinity on salt concentration, ranging from 20 mM to 150 mM (physiological concentration) salt. We found that the binding affinity of some RNAs demonstrate a linear dependence on ionic strength, while other ligands seem to be minimally affected by salt concentration. The predicted secondary structures of the collagen mRNA ligands that were minimally affected by salt indicate that HsLARP6 recognizes RNA ligands at least in part based on structure as well as sequence.